ABSTRACT Prions are infectious, self-propagating protein aggregates that were first described in the context of the transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative diseases that afflict humans and other mammals. The culprit in the case of the TSEs is an endogenous protein called PrP that has an inherent ability to undergo a dramatic conformational conversion, leading to the formation of distinctive cross-b aggregates (termed amyloid) that are both self-templating and infectious. Prions have also been uncovered in budding yeast and other fungi, where they act as protein-based genetic elements that confer new heritable phenotypes on those cells that harbor them. Like PrP, fungal prion proteins can access alternative conformational states, a soluble form and a self-perpetuating, amyloid form (the prion form) that is infectious. Unlike PrP-based prions, however, fungal prions do not typically cause cell death; they can, in fact, enhance cell survival under specific stress conditions. The foundation for the proposed studies is our discovery that prion proteins also exist in bacteria. Our research goals are to investigate the scope of prion-like phenomena in bacteria and to probe the physiologic significance of prions in bacteria. An overarching hypothesis informing our work is that protein-based heredity can serve as an epigenetic source of phenotypic diversity in the bacterial domain of life. As well as using E. coli cells as a model in which to study prion proteins from diverse bacteria, we are extending our studies to encompass species that naturally contain prion proteins, including constituents of the human microbiota. A major methodological focus of our long-term research program has been the development of broadly applicable genetic assays, and our work here includes the development and implementation of bacteria-based genetic assays that can detect prion conversion events. These genetic tools will enable screening for prion protein encoded in bacterial (and other) genomes, facilitating the discovery of new prion proteins and potentially also new classes of prion proteins. At the same time, our tools provide facile methods for addressing mechanistic questions about prion formation and prion propagation. Our approach to understanding prion biology is multi-faceted, encompassing ongoing collaboration with structural biologists and biophysicists. Because prion proteins in all domains of life share fundamental properties, what we learn in bacteria could provide mechanistic insight relevant to prion proteins in other settings; furthermore, our bacteria- based tools can be used to investigate the behavior of bacterial and non-bacterial prion proteins alike. Bacterial prions could have far-reaching human health implications; for example, as a source of non-genetic phenotypic heterogeneity, prions might enhance bacterial fitness in a pathogenic context. Moreover, the presence of prions in the human microbiota could potentially impact human health via cross-Kingdom templating interactions involving disease-associated human proteins.